Hybrid oligonucleotide phosphorothioates

ABSTRACT

The invention provides hybrid oligonucleotides having phosphorothioate or phosphorodithioate internucleotide linkages, and both deoxyribonucleosides and ribonucleosides or 2′-substituted ribonucleosides. Such hybrid oligonucleotides have superior properties of duplex formation with RNA, nuclease resistance, and RNase H activation.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to synthetic oligonucleotides that areuseful for studies of gene expression and in the antisenseoligonucleotide therapeutic approach. More particularly, the inventionrelates to synthetic oligonucleotides that have improved qualities forsuch applications resulting from modifications in the sugar phosphatebackbone of the oligonucleotides.

[0003] 2. Summary of the Related Art

[0004] The potential for the development of an antisense oligonucleotidetherapeutic approach was first suggested in three articles published in1977 and 1978. Paterson et al., Proc. Natl. Acad. Sci. USA 74: 4370-4374(1977) discloses that cell-free translation of mRNA can be inhibited bythe binding of an oligonucleotide complementary to the mRNA. Zamecnikand Stephenson, Proc. Natl. Acad. Sci. USA 75: 280-284 and 285-288(1978) discloses that a 13-mer synthetic oligonucleotide that iscomplementary to a part of the Rous sarcoma virus (RSV) genome inhibitsRSV replication in infected chicken fibroblasts and inhibitsRSV-mediated transformation of primary chick fibroblasts into malignantsarcoma cells.

[0005] These early indications that synthetic oligonucleotides can beused to inhibit virus propagation and neoplasia have been followed bythe use of synthetic oligonucleotides to inhibit a wide variety ofviruses. Goodchild et al., U.S. Pat. No. 4,806,463 (the teachings ofwhich are hereby incorporated by reference) discloses inhibition ofhuman immunodeficiency virus (HIV) by synthetic oligodeoxynucleotidescomplementary to various regions of the HIV genome. Leiter et al., Proc.Natl. Acad. Sci. USA 87: 3430-3434 (1990) discloses inhibition ofinfluenza virus by synthetic oligonucleotides. Agris et al..Biochemistry 25: 6268-6275 (1986) discloses the use of syntheticoligonucleotides to inhibit vesicular stomatitis virus (VSV). Gao etal., Antimicrob. Agents Chem. 34 808-812 (1990) discloses inhibition ofherpes simplex virus by synthetic oligonucleotides. Birg et al., NucleicAcids Res. 18: 2901-2908 (1990) discloses inhibition of simian virus(SV40) by synthetic oligonucleotides. Storey et al., Nucleic Acids Res.19: 4109-4114 (1991) discloses inhibition of human papilloma virus (HPV)by synthetic oligonucleotides. The use of synthetic oligonucleotides andtheir analogs as antiviral agents has recently been extensively reviewedby Agrawal, Trends in Biotech 10: 152-158 (1992).

[0006] In addition, synthetic oligonucleotides have been used to inhibita variety of non-viral pathogens, as well as to selectively inhibit theexpression of certain cellular genes. Thus, the utility of syntheticoligonucleotides as agents to inhibit virus propagation, propagation ofnon-viral pathogens and selective expression of cellular genes has beenwell established. However, there is a need for improved oligonucicotidesthat have greater efficacy in inhibiting such viruses, pathogens andselective gene expression. Various investigators have attempted to meetthis need by preparing and testing oligonucleotides having modificationsin their internucleoside linkages. Several investigations have shownthat such modified oligonucleotides are more effective than theirunmodified counterparts. Sarin et al., Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988) teaches that oligodeoxynucleoside methylphosphonatesare more active as inhibitors of HIV-1 than conventionaloligodeoxynucieotides. Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079-7083 (1988) teaches that oligonucleotide phosphorothioates andcertain oligonucleotide phosphoramidates are more effective atinhibiting HIV-1 than conventional oligodeoxynucleotides. Agrawal etal., Proc. Natl. Acad. Sci. USA 86: 7790-7794 (1989) discloses theadvantage of oligonucleotide phosphorothioates in inhibiting HIV-1 inearly and chronically infected cells.

[0007] In addition, chimeric oligonucleotides having more than one typeof internucleoside linkage within the oligonucleotide have beendeveloped. Chimeric oligonucleotides contain deoxyribonucleosides only,but have regions containing different internucleoside linkages Pedersonet al., U.S. Pat. No. 5,149,797, the teachings of which are herebyincorporated by reference, discloses chimeric oligonucleotides having anoligonucleotide phosphodiester or oligonucleotide phosphorothioate coresequence flanked by ollgonucleotide, methylphosphonates orphosphoramidates. Furdon et al., Nucleic Acids Res. 17: 9193-9204 (1989)discloses chimeric oligonucleotides having regions of oligonucleotidephosphodiesters in addition to either oligonucleotide phosphorothioateor methylphosphonate regions. Quartin et al., Nucleic Acids Res. 17-7523-7562 (1989) discloses chimeric oligonucleotides having regions ofoligonucleotide phosphodiesters and oligonucleotide methylphosphonates.Each of the above compounds contains deoxyribonucleotidephosphorothioates, which have reduced duplex stability. Atabekov et al.,FEBS Letters 232: 96-98 (1988) discloses chimeric oligonucleotides inwhich all internucleoside linkages are phosphodiester linkages, but inwhich regions of ribonucleotides and deoxyribonucleotides are mixed.Inoue et al., FEBS Letters, 215: 237-250 (1987) discloses chimericoligonucleotides having only phosphodiester linkages, and regions ofdeoxyribonucleotides and 2′-OMe-ribonucleotides. None of these compoundshaving solely phosphodiester linkages exhibit either endonuclease orexonuclease resistance.

[0008] Many of these modified oligonucleotides have contributed toimproving the potential efficacy of the antisense oligonucleotidetherapeutic approach. However, certain deficiencies remain in the knownoligonucleotides, and these deficiencies can limit the effectiveness ofsuch oligonucleotides as therapeutic agents. Wickstrom, J. Biochem.Biophys. Methods 13: 97-102 (1986) teaches that oligonucleotidephosphodiesters are susceptible to nuclease-mediated degradation. Suchnuclease susceptibility can limit the bioavailability ofoligonucleotides in vivo. Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401-1405 (1990) teaches that oligonucleotide phosphoramidates ormethylphosphonates when hybridized to RNA do not activate RNase H, theactivation of which can be important to the function of antisenseoligonucleotides. Agrawal et al., Nucleosides & Nucleotides 8: 5-6(1989) teaches that oligodeoxyribonucleotide phosphorothioates havereduced duplex stability when hybridized to RNA.

[0009] There is, therefore, a need for improved oligonucleotides thatovercome the deficiencies of oligonucleotides that are known in the art.Ideally, such oligonucleotides should be resistant to nucleolyticdegradation, should form stable duplexes with RNA, and should activateRNase H when hybridized with RNA.

BRIEF SUMMARY OF THE INVENTION

[0010] The invention provides hybrid oligonucleotides (containingsegments of deoxy- and ribonucleotides) that resist nucleolyticdegradation, form stable duplexes with RNA or DNA, and activate RNase Hwhen hybridized with RNA. Oligonucleotides according to the inventionprovide these features by having phosphorothioate and/orphosphorodithioate internucleoside linkages and segments ofoligodeoxyribonucleotides as well as segments of eitheroligoribonucleotides or 2′-substituted-oligoribonucleotides. Forpurposes of the invention, the term “2′-substituted” means substitutionof the 2′-OH of the ribose molecule with, -O-lower alkyl containing 1-6carbon atoms, aryl or substituted aryl or allyl having 2-6 carbon atomse.g., 2′-OMe, 2′-O-allyl, 2′-O-aryl, 2′-O-alkyl, 2′-halo, or 2′-amino,but not with 2′-H, wherein allyl, aryl, or alkyl groups may beunsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl,cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or aminogroups.

[0011] An object of the invention is to provide oligonucleotides thatcan be used to analyze and explain the importance to the effectivenessof antisense oligonucleotides of the parameters of nuclease resistance,duplex stability and RNase H activation. Another object of the inventionis to provide oligonucleotides that are effective for regulatingcellular, pathogen, or viral gene expression at the mRNA level. Yetanother object of the invention is to provide therapeuticoligonucleotides that have great efficacy in the antisenseoligonucleotide therapeutic approach. Oligonucleotides according to theinvention are useful in satisfying each of these objects of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows ion exchange HPLC analysis of nuclease treatedoligonucleotides. In FIG. 1A, profiles A, B and C arc ofoligonucleotidcs F, C and A (SEQ ID NOS:6,3 and 1, respectively),respectively after 420 minutes SVPD digestion: In FIG. 1B, profile A isof an undigested oligonucleotide phosphodiester and profile B is of thesame after 1 minute SVPD digestion (SVPD concentration one tenth that inFIG. 1A).

[0013]FIG. 2 shows results of RNase H activation studies foroligonucleotides (SEQ ID NOS:1-3 and 5-8), as described in Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In a first aspect, the invention provides oligonucleotides thatare useful for studying the parameters that are important for effectiveantisense oligonucleotide action. For purposes of the invention, theterm oligonucleotide includes polymers of two or more ribonucleotides,deoxyribonucleotides, or both, with ribonucleotide and/ordeoxyribonucleotide monomers being connected together via 5′ to 3′linkages which may include any of the linkages that are known in theantisense oligonucleotide art. In addition, the term oligonucleotidesincludes such molecules having modified nucleic acid/bases and/orsugars, as well as such molecules having added substituents, such asdiamines, cholesteryl or other lipophilic groups. Certain preferredcombinations of monomers and inter-monomer linkages are discussed ingreater detail below.

[0015] It is generally believed that the activity of an antisenseoligonucleotide depends on the binding of the oligonucleotide to thetarget nucleic acid, thus disrupting the function of the target, eitherby hybridization arrest or by destruction of target RNA by RNase H.These mechanisms of action suggest that two parameters should beimportant to antisense oligonucleotide activity: duplex stability andRNase H activation. Duplex stability is important, since theoligonucleotide presumably must form a duplex (or triplex in theHoogsteen pairing mechanism) with the target nucleic acid to act eitherby hybridization arrest or by RNase H-mediated target destruction. RNaseH activation (the ability to activate RNase H when hybridized withtarget RNA) is implicated when the target nucleic acid is RNA, sincesuch activation can lead to the effective destruction of the target RNAmolecule. In addition, for an antisense oligonucleotide to act in vivo,it must survive long enough to interact with the target nucleic acid.Given the fact that the in vivo environment contains endonuclease andcxonuclease activities, a third parameter arises from this requirement;namely that the antisense oligonucleotide should resist nuclcolyticdegradation.

[0016] To analyze and explain the importance of each of these parametersto the effectiveness of antisense oligonucleotides, it is necessary tohave oligonucleotides that vary in each of these parameters. Theproperties of several known oligonucleotides arc shown in Table I,below. TABLE I PROPERTIES OF OLIGONUCLEOTIDES Duplex Nuclease RNase HOligonucleotide Stability¹ Resistance² Activation³Oligodeoxyribonucleotide — — Yes (phosphate) OligodeoxyribonucleotideLower + Yes (phosphorothioate) Oligodeoxyribonucleotide Lower ++ Yes(phosphorodithioate) Oligodeoxyribonucleotide Lower + N.K. (selenoate)Oligodeoxyribonucleotide Lower +++ No (phosphoramidate)Oligoribonucleotide Higher — No (phosphate) OligodeoxyribonucleotideHigher + No (phosphorothioate) 2′-OMe-Oligonucleotide Higher + No(phosphate) 2′-OMe-Oligonucleotide Higher ++ No (phosphorothioate)Oligodeoxyribonucleotide Lower +++ No (methylphosphonate)

[0017] Hybrid oligonucleotides according to the invention form morestable duplexes with complementary RNA than oligodeoxyribonucleotidephosphorothioates. In addition, they are more resistant toendonucleolytic and exonucleolytic degradation thanoligodeoxyribonucleotide phosphorothioates and they normally activateRNase H. Consequently, oligonucleotides according to the inventioncomplement the oligonucleotides shown in Table I in studies of theparameters involved in the effectiveness of antisense oligonucleotides.

[0018] With respect to this first aspect of the invention,oligonucleotides according to the invention can have any oligonucleotidesequence, since complementary oligonucleotides used in such study can beprepared having any oligonucleotide sequence. Oligonucleotides accordingto this aspect of the invention are characterized only by the followingfeatures. First, at least some of the internucleoside linkages presentin oligonucleotides according to the invention are phosphorothioateand/or phosphorodithioate linkages. In various embodiments, the numberof phosphorothioate and/or phosphorodithioate internucleotide linkagescan range from 1 to as many internucleotide linkages as are present inthe oligonucleotide. Thus, for purposes of the invention, the termoligonucleotide phosphorothioate and/or phosphorodithioate is intendedto encompass every such embodiment. In a preferred embodiment,oligonucleotides according to the invention will range from about 2 toabout 50 nucleotides in length, and most preferably from about 6 toabout 50 nucleotides in length. Thus, in this preferred embodiment,oligonucleotides according to the invention will have from 1 to about 49phosphorothioate and/or phosphorodithioate internucleotide linkages.

[0019] A second feature of oligonucleotides according to this aspect ofthe invention is the presence of deoxyribonucleotides. Oligonucleotidesaccording to the invention contain at least one deoxyribonucleotide.Preferably oligonucleotides according to the invention contain four ormore deoxyribonucleotides in a contiguous block, so as to provide anactivating segment for RNase H. In certain preferred embodiments, morethan one such activating segment will be present. Such segments may bepresent at any location within the oligonucleotide. There may be amajority of deoxyribonucleotides in oligonucleotides according to theinvention. In fact, such oligonucleotides may have as many as all butone nucleotide being deoxyribonucleotides. Thus, in a preferredembodiment, having from about 2 to about 50 nucleotides or mostpreferably from about 6 to about 50 nucleotides, the number ofdeoxyribonucleotides present will range from 1 to about 49deoxyribonucleotides.

[0020] A third feature of oligonucleotides according to this aspect ofthe invention is the presence of ribonucleotides, 2′-substitutedribonucleotides or combinations thereof. For purposes of the invention,the term “2′-substituted” means substitution of the 2′-OH of the ribosemolecule with, e.g. 2′-OMe, 2′-O-allyl, 2′-O-aryl, 2′-O-alkyl, 2′-halo,or 2′-amino, but not with 2′-H, wherein allyl, aryl, or alkyl groups maybe unsubstituted or substituted, e.g. with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl or amino groups. Oligonucleotides according to the inventioncontain at least one ribonueleotide and/or 2′-substitutedribonucleotide. In a preferred embodiment, such oligonucleotides have 6or more ribonucleotides and/or 2′-substituted ribonucleotides to enhanceduplex stability, Such ribonucleotides and/or 2′-substitutedribonucleotides can be present singly, in pairs, or in larger contiguoussegments, and may be present at any position within the oligonucleotideor at multiple positions within the oligonucleotide. Suchribonucleotides and/or 2′-substituted ribonucleotides may comprise asmany as all but one nucleoside within the oligonucleotides. Thus, in apreferred embodiment, having from about 2 to about 50 nucleotides ormost preferably from about 6 to about 50 nucleotides, the number ofribonucleosides or 2′-substituted ribonucleotides will range from about1 to about 49 deoxyribonucleotides.

[0021] The ability to vary the numbers and positions of phosphorothioateand/or phosphorodithioate internucleotide linkages,deoxyribonucleotides, and ribonucleotides or 2′-substitutedribonucleotides allows the investigator to examine in detail how each ofthese variables affects the parameters of nuclease resistance, duplexstability and RNase H activation. The ability to vary the size of theoligonucleotide allows examination of yet another parameter. Inaddition, smaller oligos (e.g., dimers) can be used as building blocksfor larger oligos. Thus, the embodiments described above are useful insuch studies.

[0022] In a second aspect, the invention provides hybridoligonucleotides that are effective in inhibiting viruses, pathogenicorganisms, or the expression of cellular genes. The ability to inhibitsuch agents is clearly important to the treatment of a variety ofdisease states. Oligonucleotides according to this aspect of theinvention share the characteristics of the above-describedoligonucleotides, except that the oligonucleotide sequence ofoligonucleotides according to this aspect of the invention iscomplementary to a nucleic acid sequence that is from a virus, apathogenic organism or a cellular gene. Preferably such oligonucleotidesare from about 6 to about 50 nucleotides in length. For purposes of theinvention, the term “oligonucleotide sequence that is complementary to anucleic acid sequence” is intended to mean an oligonucleotide sequence(2 to about 50 nucleotides) that binds to the nucleic acid sequenceunder physiological conditions, e.g. by Watson-Crick base pairing(interaction between oligonucleotide and single-stranded nucleic acid)or by Hoogsteen base pairing (interaction between oligonucleotide anddouble-stranded nucleic acid) or by any other means including in thecase of a oligonucleotide binding to RNA, pseudoknot formation. Suchbinding (by Watson Crick base pairing) under physiological conditions ismeasured as a practical matter by observing interference with thefunction of the nucleic acid sequence.

[0023] The nucleic acid sequence to which an oligonucleotide accordingto the invention is complementary will vary, depending upon the agent tobe inhibited. In many cases the nucleic acid sequence will be a virusnucleic acid sequence. The use of antisense oligonucleotides to inhibitvarious viruses is well known, and has recently been reviewed inAgrawal, Trends in Biotech 10:152-158 (1992). Viral nucleic acidsequences that are complementary to effective antisense oligonucleotideshave been described for many viruses, including human immunodeficiencyvirus type 1 (U.S. Pat. No. 4,806,463, the teachings of which are herebyincorporated by reference), herpes simplex virus (U.S. Pat. No.4,689,320, the teachings of which are hereby incorporated by reference),influenza virus (U.S. Pat. No. 5,194,428, the teachings of which arehereby incorporated by reference), and human papilloma virus (Storey etal., Nucleic Acids Res. 19:4109-41 14 (1991)). Sequences complementaryto any of these nucleic acid sequences can be used for oligonucleotidesaccording to the invention, as can be oligonucleotide sequencescomplementary to nucleic acid sequences from any other virus. Additionalviruses that have known nucleic acid sequences against which antisenseoligonucleotides can be prepared include foot and mouth disease virus(See Robertson et al., J. Virology 54: 651 (1985); Harris et al., J.Virology 36: 659 (1980)), yellow fever virus (See Rice et al., Science229: 726 (1985)), varicella-zoster virus (See Davison and Scott, J. Gen.Virology 67: 2279 (1986), and cucumber mosaic virus (See Richards etal., Virology 89: 395 (1978)).

[0024] Alternatively, oligonucleotides according to the invention canhave an oligonucleotide sequence complementary to a nucleic acidsequence of a pathogenic organism. The nucleic acid sequences of manypathogenic organisms have been described, including the malariaorganism, Plasmodium falciparum, and many pathogenic bacteria.Oligonucleotide sequences complementary to nucleic acid sequences fromany such pathogenic organism can be used in oligonucleotides accordingto the invention. Examples of pathogenic eukaryotes having known nucleicacid sequences against which antisense oligonucleotides can be preparedinclude Trvpanosom abrucei gambiense and Leishmania (See Campbell etal., Nature 311: 350 (1984)), Fasciola hepatica (See Zurita et al.,Proc. Natl. Acad. Sci. USA 84: 2340 (1987). Antifungal oligonucleotidescan be prepared using a target hybridizing region having anoligonucleotide sequence that is complementary to a nucleic acidsequence from, A, the chitin synthetase gene, and antibacterialoligonucleotides can be prepared using, e.g., the alanine racemase gene.

[0025] In yet another embodiment, the oligonucleotides according to theinvention can have an oligonucleotide sequence complementary to acellular gene or gene transcript, the abnormal expression or product ofwhich results in a disease state. The nucleic acid sequences of severalsuch cellular genes have been described, including prion protein (Stahland Prusiner, FASEB J. 5: 2799-2807 (1991)), the amyloid-like proteinassociated with Alzheimer's disease (U.S. Pat. No. 5,015,570, theteachings of which are hereby incorporated by reference), and variouswell-known oncogenes and proto-oncogenes, such as c-myb, c-myc, c-abland n-ras. In addition, oligonucleotides that inhibit the synthesis ofstructural proteins or enzymes involved largely or exclusively inspermatogenesis, sperm motility, the binding of the sperm to the egg orany other step affecting sperm viability may be used as contraceptives.Similarly, contraceptives for women may be oligonucleotides that inhibitproteins or enzymes involved in ovulation, fertilization, implantationor in the biosynthesis of hormones involved in those processes.

[0026] Hypertension may be controlled by oligodeoxynucleotides thatsuppress the synthesis of angiotensin converting enzyme or relatedenzymes in the renin/angiotensin system; platelet aggregation may becontrolled by suppression of the synthesis of enzymes necessary for thesynthesis of thromboxane A2 for use in myocardial and cerebralcirculatory disorders, infarcts, arteriosclerosis, embolism andthrombosis; deposition of cholesterol in arterial wall may be inhibitedby suppression of the synthesis of fatty acid co-enzyme A; cholesterolacyl transferase in arteriosclerosis; inhibition of the synthesis ofcholinephosphotransferase may be useful in hypolipidemia.

[0027] There are numerous neural disorders in which hybridization arrestmay be used to reduce or eliminate adverse effects of the disorder. Forexample, suppression of the synthesis of monoamine oxidase may be usedin Parkinson's disease; suppression of catechol o-methyl transferase maybe used to treat depression; and suppression of indole N-methyltransferase may be used in treating schizophrenia.

[0028] Suppression of selected enzymes in the arachidonic acid cascadewhich leads to prostaglandins and leukotrienes may be useful in thecontrol of platelet aggregation, allergy, inflammation, pain and asthma.

[0029] Suppression of the protein expressed by the multidrug resistance(mdr) gene, which can be responsible for development of resistance oftumors to a variety of anti-cancer drugs and is a major impediment inchemotherapy may prove to be beneficial in the treatment of cancer.Oligonucleotide sequences complementary to nucleic acid sequences fromany of these genes can be used for oligonucleotides according to theinvention, as can be oligonucleotide sequences complementary to anyother cellular gene or gene transcript, the abnormal expression orproduct of which results in a disease state.

[0030] Antisense regulation of gene expression in plant cells has beendescribed in U.S. Pat. No. 5,107,065, the teachings of which are herebyincorporated by reference.

[0031] In a third aspect, the invention provides therapeuticpharmaceutical formulations of oligonucleotides that are effective fortreating virus infection, infections by pathogenic organisms, or diseaseresulting from abnormal gene expression or from the expression of anabnormal gene product. Such therapeutic pharmaceutical formulationscomprise the oligonucleotides according to the second aspect of theinvention in a pharmaceutically acceptable carrier.

[0032] In a fourth aspect, the invention provides a method forinhibiting the gene expression of a virus, a pathogenic organism or acellular gene, the method comprising the step of providingoligonucleotides according to the invention to cells infected with thevirus or pathogenic organism in the former two cases or to cellsgenerally in the latter case. Such methods are useful in studying geneexpression and the function of specific genes.

[0033] In a fifth aspect, the invention provides a method of treating adiseased human or animal in which the disease results from infectionwith a virus or pathogenic organism, or from the abnormal expression orproduct of a cellular gene. The method comprises administeringtherapeutic pharmaceutical formulations of oligonuclcotides according tothe invention to the diseased human or animal. Preferably, the routes ofsuch administration will include oral, intranasal, rectal and topicaladministration. In such methods of treatment according to the inventionthe oligonucleotides may be administered in conjunction with othertherapeutic agents, e.g., AZT in the case of AIDS.

[0034] A variety of viral diseases may be treated by the method oftreatment according to the invention, including AIDS, ARC, oral orgenital herpes, papilloma warts, flu, foot and mouth disease, yellowfever, chicken pox, shingles, adult T cell-leukemia, and hepatitis.Among fungal diseases that may be treatable by the method of treatmentaccording to the invention are candidiasis, histoplasmosis,cryptococcocis, blastomycosis, aspergillosis, sporotrichosis,chromomycosis, dermatophytosis and coccidioidomycosis. The method mightalso be used to treat rickettsial diseases e.g., typhus, Rocky Mountainspotted fever), as well as sexually transmitted diseases caused byChlamydia trachomatis or Lymnhogranuloma venereum. A variety ofparasitic diseases may be treated by the method according to theinvention, including amebiasis, Chegas' disease, toxoplasmosis,pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, andPneumocystis carini pneumonia; also worm (helminthic) diseases such asascariasis, filariasis, trichinosis, schistosomiasis and nematode orcestode infections. Malaria may be treated by the method of treatment ofthe invention regardless of whether it is caused by P. falciparum, P.vivax, P. orale, or P. malariae.

[0035] The infectious diseases identified above may all be treated bythe method of treatment according to the invention because theinfectious agents for these diseases are known and thus oligonucleotidesaccording to the invention can be prepared, having oligonucleotidesequence that is complementary to a nucleic acid sequence that is anessential nucleic acid sequence for the propagation of the infectiousagent, such as an essential gene.

[0036] Other disease states or conditions that may be treatable by themethod according to the invention are those which result from anabnormal expression or product of a cellular gene. These conditions maybe treated by administration of oligonucleotides according to theinvention, and have been discussed earlier in this disclosure.

[0037] Oligonucleotides according to the invention can be synthesized byprocedures that are well known in the art. Alternatively, and preferablysuch oligonucleotides can be synthesized by the H-phosphonate approachdescribed in U.S. Pat. No. 5,149,798, the teachings of which are herebyincorporated by reference, and in Agrawal and Tang, Tetrhadron Lett. 31:7541-7544 (1990). Oligonucleotides according to the invention can bemade even more resistant to nucleolytic degradation through the additionof cap structures at the 5′ and/or 3′ end.

[0038] The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in nature.

EXAMPLE 1 Synthesis of Hybrid Oligonucleotide Phosphorothioates

[0039] Hybrid oligonucleotide phosphorothioates were synthesized on CPGon a 5-6 μmole scale on an automated synthesizer (model 8700, Millipore,Milford, Mass.) using the H-phosphonate approach described in U.S. Pat.No. 5,149,798. Deoxynucleoside H-phosphonates were obtained fromMillipore. 2′-OMe ribonucleotide H-phosphonates were synthesized bystandard procedures. Segments of oligonucleotides containing 2′-OMenucleoside were assembled by using 2′-OMe ribonucleoside H-phosphonatesfor the desired cycles. Similarly, segments of oligonucleotidescontaining deoxyribonucleosides were assembled by using deoxynucleosideH-phosphonates for the desired cycles. After assembly, CPG boundoligonucleotide H-phosphonate was oxidized with sulfur to generate thephosphorothioate linkage. Oligonucleotides were then deprotected inconcentrated NH,OH at 40° C. for 48 hours.

[0040] Crude oligonucleotide (about 500 A₂₈₀ units) was analyzed onreverse low pressure chromatography on a C₁₈ reversed phase medium. TheDMT group was removed by treatment with 80% aqueous acetic acid, thenthe oligonucleotides were dialyzed against distilled water andlyophilized.

[0041] The oligonucleotides synthesized are. shown in Table 11, below.Oligonucleotides A-correspond to SEQ ID NOS:1-6, respectively. TABLE IIHYBRID OLIGONUCLEOTIDE PHOSPHOROTHIOATES' SYNTHESIZED Oligo Structure A^(5′)A C A C C C A A T T C T G A A A A T G G^(3′) B A C A C C C A A T TC U\G\A\A\A\A\U\G G C A\C\A C C C A A T T C T G A A A\A\U\G G D A\C A CC C\A A T T C U\G A A A A U\G G E A C\A\C\C\C\A\A\U T C T G A A A A T GG F A\C\A\C\C\C\A\A\U\U\C\U\G\A\A\A\A\U\G G

EXAMPLE 2 Relative Nuclease Resistance of Hybrid OligonucleotidePhosphorothioates

[0042] To test the relative nuclease resistance of various hybridoligonucleotide phosphorothioates, the oligonucleotides were treatedwith snake venom phosphodiesterase (SVPD). About 0.2 A₂₈₀ units ofoligos A, C and F were dissolved in 500 μl buffer (40 mM NH₄CO₃, pH0.4+20 mM MgCl₂) and mixed with 0.1 units SVPD. The mixture wasincubated at 37° C. for 420 minutes. After 0, 200 and 420 minutes, 165μl aliquots were removed and analyzed using ion exchange HPLC. Theresults are shown in FIG. 1. Oligonucleotide F (SEQ ID NO:6) was veryresistant to phosphodiesterase, whereas oligonucleotide A (SEQ ID NO:1)was digested almost to completion and oligonucleotide C (SEQ ID NO:3)was digested to 50% (panel A). An oligonucleotide phosphodiester wasdigested to about 80% in one minute using one tenth of the concentrationof SVPD. (panel B)

[0043] These results indicate that the presence of 2′-OMeribonucleotides in an oligonucleotide phosphorothioate enhancesresistance to exonucleolytic digestion and that this enhanced resistanceincreases when a larger proportion of 2′-OMe ribonucleotides are used.Due to the similar character and behavior of ribonucleotides, other2′-substituted ribonucleotides and 2′-OMe ribonucleotides, these resultsalso suggest that similar enhancement of nuclease resistance would beobtained for hybrid oligonucleotide phosphorothioates and/orphosphorodithioates having ribonucleotides, 2′-substitutedribonucleotides, or a mixture of ribonucleotides and 2′-substitutedribonucleotides.

EXAMPLE 3 Relative Duplex Stability of Hybrid OligonucleotidePhospohorothioates

[0044] Oligonucleotides A-F (SEQ ID NOS:1-6) were tested for therelative stability of duplexes formed between them and complementaryoligodeoxyribonucleotides, and with complementary oligoribonucleotides.In separate reactions, each oligonucleotide A-F (SEQ ID NOS:1-6) wasmixed with an equivalent quantity (0.2 A₂₈₀ units) of its complementaryoligonucleotide in 150 mM NaCl, 10 mM Na₂PO₄, 1 mM EDTA, pH 7. Themixture was heated to 85° C. for 5 minutes, then cooled to 30° C. Thetemperature was then increased from 30° C. to 80° C. at a rate of 1° C.per minute and A₂₈₀ was recorded as a function of temperature. Theresults are shown in Table III, below, where oligonucleotides A-Fcorrespond to SEQ ID NOS:1-6, respectively. TABLE III MELTINGTEMPERATURE OF DUPLEXES Tm DNA-RNA Duplex-Tm DNA DNA-RNA DuplexOligonucleotide DNA-DNA Duplex DNA-RNA Duplex Duplex (° C.) w/MagnesiumDifferences Differences Difference in Tm in Tm in Tm compared comparedcompared to to oligo- to oligo- oligo- Tm DNA nucleotide A Tm RNAnucleotide A Tm RNA nucleotide A (° C.) (° C.) (° C.) (° C.) (° C.) (°C.) A 51.1 0 43.4 0 −7.7 48.1 0 B 48.3 −2.8 50.9 7.5 2.6 58.4 10.3 C49.9 −1.2 48.9 5.5 −1.0 54.2 6.1 D 45.1 −6.0 50.9 7.5 5.8 56.1 8.0 E47.2 −3.9 51.1 7.7 3.9 56.5 8.4 F 47.6 −3.5 61.1 17.7 13.5 69.1 21.0

[0045] These results reveal that when the complementary oligonucleotideis an oligoribonucleotide, the presence of 2′-OMe ribonucleotidesenhances duplex stability, and that this enhancement increases withincreased proportions of 2′-OMe ribonucleotides. These results should besimilarly applicable to hybrid oligonucleotide phosphorothioates and/orphosphorodithioates containing ribonucleotides, 2′-substitutedribonucleotides, or mixtures of ribonucleotides and 2′-substitutedribonucleotides. Thus, the hybrid oligonucleotide phosphorothioatesand/or phosphorodithioates according to the invention should bind viralRNA or virus, pathogenic organism or cellular mRNA with greater affinitythan ordinary oligodeoxynucleotide phosphorothioates.

EXAMPLE 4 Activation of RNase H by Hybrid OligonucleotldePhosphorothioates

[0046] Oligonucleotide phosphorothioates and various hybridoligonucleotide phosphorothioates were studied for their RNase Hactivation properties. Oligonucleotide A (SEQ ID NO:1) (Table II), anoligonucleotide phosphorothioate which is known to activate RNase H, wasused as a control. oligonucleotide F (SEQ ID NO:6) (a 2′-OMe analog ofoligonucleotide phosphorothioate) and oligonucleotides C, B, and E (SEQID NOS:3,2 and 5, respectively), hybrid oligonucleotides, were studiedfor their ability to activate RNase H.

[0047] To carry out the experiment, a complementary 32-meroligoribonucleotide was synthesized (FIG. 2) and kinased at the 5′-end,³²P-labeled 32-mer RNA (0.003 A₂₈₀ units; 0.01 μg) and oligonucleotides(0.0635 A₂₈₀ units; 1.9 μg) were mixed in the 20 μl of buffer (0.15 MNaCl, 0.01 MgCl₂ 0.01 M Tris-HCI, pH 7.9, containing 0.001 M DTT. Themixture was incubated with 6 units of RNase H (E. coli) at 37° C.Aliquots of 4.5 μl were removed at 0, 15, 30, and 60 minutes andanalyzed on polyacrylamide gel electrophoresis.

[0048] Oligonucleotide A (SEQ ID NO:1) (Duplex A in FIG. 2) showed sitespecific cleavage of RNA by RNase H. Oligonucleotide F (SEQ ID NO:6)(2′-OMe analog; Duplex B) showed no cleavage of RNA in presence of RNaseH. Hybrid oligonucleotide B, C, and E (SEQ ID NOS:2, 3 and 5,respectively) (Duplexes C, D, and E, respectively) showed site specificcleavage of RNA by RNase H. Duplex F, in which a mismatchedoligonucleotide phosphorothioate (SEQ ID NO:7) was studied showed nocleavage of RNA. Lane G shows that in presence of RNase H, RNA was notcleaved.

EXAMPLE 5 Inhibition of HIV by Hybrid Oligonucleotide Phosphorothioates

[0049] Hybrid oligonucleotide phosphorothioates were tested for theirability to inhibit HIV-1 in tissue culture. H9 lymphocytes were infectedwith HIV-1 virions (=0.01-0.1 TCID₉₀/cell) for one hour at 37° C. Afterone hour, unadsorbed virions were washed and the infected cells weredivided among wells of 24 well plates. To the infected cells, anappropriate concentration (from stock solution) of oligonucleotide wasadded to obtain the required concentration in 2 ml medium. The cellswere then cultured for four days. At the end of four days, level ofHIV-1 expression was measured by synthetic formation, p24 expression andreverse transcriptase activity. The level of expression of p24 wascompared between oligonucleotide treated and untreated (no drug)infected cells. (Table IV)

[0050] All of the hybrid oligonucleotide phosphorothioates tested showedsignificant inhibition of synthetic formation, p24 expression andreverse transcriptase, without significant cytotoxicity. These resultsindicate that hybrid oligonucleotide phosphorothioates containing 2′-OMeribonucleotides are more effective as inhibitors of gene expressioncompared to oligodioxynucleotide phosphoration. Similar effectivenesswould be expected for hybrid oligonucleotide phosphorothioates and/orphosphorodithioates containing ribonucleosides, 2′-substitutedribonucleosides, or a mixture of ribonucleosides and 2′-substitutedribonucleosides. The activity of compounds listed in Table III arelisted in Table IV below, where oligonucleotides A-F correspond to SEQID NOS:1-6, respectively. TABLE IV Anti-HIV activity of oligonucleotides(listed in Table III) Inhibition of Inhibition of ReverseOligonucleotides Syncytia formed p24 (%) Transcriptase (%) Oligo A ++ 9388 Oligo B ++ 95 97 Oligo C + 98 97 Oligo D + 98 83 Oligo E +++ 90 94Oligo F +++ 88 91 +Control ++++ −Control —

[0051]

1 8 1 20 DNA Artificial Sequence Oligonucleotide phosphorothioate. 1acacccaatt ctgaaaatgg 20 2 20 DNA Artificial Sequence HybridOligonucleotide. 2 acacccaatt cugaaaaugg 20 3 20 DNA Artificial SequenceHybrid oligonucleotide. 3 acacccaatt ctgaaaaugg 20 4 20 DNA ArtificialSequence Hybrid oligonucleotide. 4 acacccaatt cugaaaaugg 20 5 20 DNAArtificial Sequence Hybrid oligonucleotide. 5 acacccaaut ctgaaaatgg 20 620 DNA Artificial Sequence 2′-OMe analog of oligonucleotidephosphorothioate. 6 acacccaauu cugaaaaugg 20 7 20 DNA ArtificialSequence Oligonucleotide. 7 acagacttac ctcagataat 20 8 32 DNA ArtificialSequence Oligonucleotide. 8 uacagcugug gguuaagacu uuuaccuauu ug 32

We claim:
 1. A hybrid oligonucleotide phosphorothioate orphosphorodithioate comprising one each of the following: adeoxyribonucleoside, a ribonucleoside or a 2′-substitutedribonucleoside, and a phosphorothioate or phosphorodithioateinternucleotide linkage.
 2. A hybrid oligonucleotide phosphorothioate orphosphorodithioate according to claim 1, wherein the deoxyribonucleotideis present in a segment of at least four contiguousdeoxyribonucleotides.
 3. A hybrid oligonucleotide phosphorothioate orphosphorodithioate according to claim 1, wherein the ribonucleotide or2′-substituted ribonucleotide is present in a segment of at least twocontiguous ribonucleotides or 2′-substituted ribonucleotides.
 4. Ahybrid oligonucleotide phosphorothioate or phosphorodithioate accordingto claim 1, having an oligonucleotide sequence that is complementary toa nucleic acid sequence from a virus, a pathogenic organism, or acellular gene.
 5. A hybrid oligonucleotide phosphorothioate orphosphorodithioate according to claim 2, having an oligonucleotidesequence that is complementary to a nucleic acid sequence from a virus,a pathogenic organism, or a cellular gene.
 6. A hybrid oligonucleotidephosphorothioate or phosphorodithioate according to claim 3, having anoligonucleotide sequence that is complementary to a nucleic acidsequence from a virus, a pathogenic organism, or a cellular gene.
 7. Atherapeutic pharmaceutical formulation comprising an oligonucleotideaccording to claim 4 in a pharmaceutically acceptable carrier.
 8. Atherapeutic pharmaceutical formulation comprising an oligonucleotideaccording to claim 5 in a pharmaceutically acceptable carrier.
 9. Atherapeutic pharmaceutical formulation comprising an oligonucleotideaccording to claim 6 in a pharmaceutically acceptable carrier.
 10. Amethod of inhibiting the gene expression of a virus, a pathogenicorganism, or a cellular gene, the method comprising the step ofproviding an oligonucleotide according to claim 4 to a cell that isinfected with a virus, to a pathogenic organism, or to a cell,respectively.
 11. A method of inhibiting the gene expression of a virus,a pathogenic organism, or a cellular gene, the method comprising thestep of providing an oligonucleotide according to claim 5 to a cell thatis infected with a virus, to a pathogenic organism, or to a cell,respectively.
 12. A method of inhibiting the gene expression of a virus,a pathogenic organism, or a cellular gene, the method comprising thestep of providing an oligonucleotide according to claim 6 to a cell thatis infected with a virus, to a pathogenic organism, or to a cell,respectively.
 13. A hybrid oligonucleotide according to claim 6 which iscomplementary to a nucleic acid sequence in a virus involved in adisease selected from the group consisting of AIDS, oral and genitalherpes, papilloma warts, influenza, foot and mouth disease, yellowfever, chicken pox, shingles, adult T-cell-leukemia and hepatitis.
 14. Ahybrid oligonucleotide according to claim 6 which inhibits theexpression of a protein in a male or female which is necessary forfertility.
 15. A hybrid oligonucleotide according to claim 6 whichinhibits the expression of a protein formed in Alzheimer disease.
 16. Ahybrid oligonucleotide according to claim 6 which inhibits expression ofa protein in a parasite causing a parasitic disease selected from thegroup consisting of amebiasis, Chegas' disease, toxoplasmosis,pneumocytosis, giardiasis, cryptoporidiosis, trichomoniasis, malaria,ascariasis, filariasis, trichinosis, schistosomiasis infections.